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Background  Cancer stem cell (CSC) is identified in osteosarcoma (OS) and considered resistant to chemotherapeutic agents. However, the mechanism of osteosarcoma stem cell (OSC) resistant to chemotherapy remains debatable and vague, and the metabolomics feature of OSC is not clarified.    Materials and methods  OSC was isolated by using sphere forming assay and identified. Untargeted LC–MS/MS analysis was performed to reveal the metabolomics feature of OSC and underlying mechanisms of OSC resistant to methotrexate (MTX).    Results  OSC was efficiently isolated and identified from human OS 143B and MG63 cell lines with enhanced chemo-resistance to MTX. The untargeted LC–MS analysis revealed that OSC showed differential metabolites and perturbed signaling pathways, mainly involved in metabolisms of fatty acid, amino acid, carbohydrate metabolism and nucleic acid. After treated with MTX, metabolomics feature of OSC was mainly involved metabolisms of amino acid, fatty acid, energy and nucleic acid. Moreover, compared with their parental OS cells response to MTX, the differential metabolites and perturbed signaling pathways were mainly involved in metabolism of amino acid, fatty acid and nucleic acid. What’s more, Rap1 signaling pathway and Ras signaling pathway were involved in OS cells and their SCs response to MTX.    Conclusion  Sphere-forming assay was able to efficiently isolate OSC from human OS cell lines and the untargeted LC–MS/MS analysis was suggested a sufficient methodology to investigate metabolomics features of OS cells and OSCs. Moreover, the metabolomics features of OSCs response to MTX might reveal a further understanding of chemotherapeutic resistance in OS.

Untargeted LC–MS/MS analysis reveals metabolomics feature of osteosarcoma stem cell response to methotrexate